PROJECT SUMMARY Mitochondria are centers of metabolism and signaling whose function is essential to all but a few eukaryotic cell types. Despite their position as the iconic powerhouses of cellular biology, many aspects of mitochondria remain remarkably obscure?a fact that contributes to our near complete inability to address mitochondrial dysfunction therapeutically. Such dysfunction is associated with a spectrum of rare inborn errors of metabolism and an increasing number of common diseases?including Parkinson?s, Alzheimer?s, various cancers, and type 2 diabetes?often through distinct means. For instance, aberrant mitochondrial biogenesis can fail to properly set cellular mitochondrial content; dysregulated signaling processes can fail to calibrate mitochondrial activity to changing cellular needs; and malfunctioning proteins can render core bioenergetic processes ineffectual. A major bottleneck to understanding?and ultimately addressing?these processes is that the proteins driving them have often not been identified. Concurrently, the functions of hundreds of known mitochondrial proteins that may fulfill these roles are undefined, or at best are poorly understood. In 2008, I led an integrative effort to generate a comprehensive compendium of the mammalian mitochondrial proteome?termed MitoCarta?that doubled the number of known mammalian mitochondrial proteins and exposed this major gap in knowledge: A striking ~300 of the ~1100 proteins had no annotated function, including ~50 that are now directly associated with human disease. Thus, the high-level goal of my research program is to achieve a more comprehensive understanding of mitochondrial biology by systematically establishing the functions of orphan mitochondrial proteins and their roles within disease-related processes. We do so by first devising novel, multi-dimensional analyses designed to make new connections between these proteins and established pathways and processes. These include customized, high-throughput protein-protein interaction screens, large- scale mass spectrometry-based profiling of yeast and human cell gene knockouts, and computational approaches. We then employ mechanistic and structural approaches to define the functions of select proteins at biochemical depth, including ancient and atypical kinases and lipid binding proteins that enable the mitochondrial coenzyme Q biosynthesis pathway and other essential metabolic processes. Finally, we investigate how post- transcriptional and post-translational regulators operate to establish a customized mitochondrial infrastructure capable of meeting changing cellular needs. Overall, by purposefully elucidating the unexplored areas of mitochondrial biology, we are rapidly arriving at a more complete understanding of what these organelles do and how their protein componentry enables their myriad functions. These efforts promise to help establish a deep, mechanistic understanding of mitochondrial biochemistry that will motivate novel therapeutic strategies for the vast array of human disorders rooted in mitochondrial dysfunction. !